The immune system is the body's defense mechanism against foreign substances and invading microorganisms. The underlying operating principle of the immune system is a self/non-self recognition pattern. If the invader organism is recognized as not being part of the "self", then a defensive immune response is mounted against it. In the case of autoimmune diseases, the immune system fails to properly recognize "self" and mounts a defense immune response against its own normal body components.
Antibodies generated by the immune system to diverse tissue and cellular components have been used to diagnose and monitor autoimmune disease activity. In systemic lupus erythematosus (SLE) (a type of autoimmune disease) one of the antibodies produced reacts with DNA that is found widely distributed in cell nuclei in a multitude of body tissues. Formation of antibodies to double-stranded or native deoxyribonucleic acid (anti-dsDNA), is relatively specific to SLE. Although other disorders, such as Mixed Connective Tissue Disease (MCTD), drug induced lupus (DIL), Rheumatoid Arthritis, Scleroderma, and Sjorgren Syndrome, produce similar clinical manifestations as SLE, high levels of anti-dsDNA are seldom associated with these disorders. Therefore, detecting anti-dsDNA is useful in specifically diagnosing SLE. Anti-dsDNA levels correlate well with the disease activity of the patient; thus making it a good monitoring tool.
Many different techniques and different diagnostic kits have been developed in the search for a standardized, accurate, rapid, and stable method for the detection of anti-dsDNA. Most of the methods have been somewhat successful, but due to unacceptably high levels of cross-reactivity (such as with single-stranded DNA [ssDNA]), the slow run time of the assays, and the short shelf life of the kit components, no method is fully adequate. Examples of the limitations of the prior methods follow.
A number of techniques have been developed to detect antibodies to dsDNA, including immunofluorescent assays (FIA), radioimmunoassay (RIA), and enzyme-linked immunosorbent assays (ELISA).
Firstly, the RIA technique has been developed in a variety of formats to measure levels of anti-dsDNA in sera. A RIA diagnostic test kit (using the Farr technique) has been developed by Diagnostic Products, Inc. for commercial sale to clinical laboratories. This test precipitates the bound labeled DNA which is then retained for counting. This test is sensitive to high levels of anti-dsDNA but has less specificity in lower levels of anti-dsDNA activity, thus resulting in false negatives. Unfortunately, this test has a run time of approximately two hours and fifteen minutes, and a limited stable shelf life. Furthermore, this test kit has the same draw-backs which are inherent in all RIAs; the expense associated with radioactive material, which also has a limited shelf life and can be potentially dangerous.
Secondly, Crithidia luciliae immunofluorescence tests have been developed as diagnostic test kits for the detection of anti-dsDNA. This test kit is based on a staining method associated with the kinetoplast of the protozoan. A positive reaction is demonstrated by detection of specific fluorescence in the kinetoplast of most cells. The kinetoplast, within the protozoan, contains many other components besides dsDNA which can cross react with anti-dsDNA or other antibodies, thus rendering false positive results. This test is extremely subjective, as it is based on an individual's ability to recognize a positive fluorescent pattern. Furthermore, this method, which has a run time of approximately two hours, has a lack of sensitivity in low levels of concentration of anti-dsDNA resulting in false negatives. The false negative and false positive results make this method more useful when used together with other tests in clinical practice.
Thirdly, the immunological community has developed diagnostic test kits for the determination of anti-dsDNA levels in the standard ELISA format. Although a variety of these kits have been developed, the same limitations of stability, cross reactivity with ssDNA, and length of run time plague each assay. Many of these kits claim a low level of cross reactivity with ssDNA which should render good sensitivity to low, medium, and high levels of anti-dsDNA; however, as evidenced by the kit instructions, the test shows a lack of sensitivity to the sera sample which are borderline positives. As a consequence, these kits cannot be used to accurately monitor patients who are borderline positives. In fact, most kits require that low positives must be retested to assure confidence in the results, thereby increasing the cost to the patient. Development of an assay which is sensitive to all levels of anti-dsDNA can only be achieved by reducing non-specific binding by such components as ssDNA.
R. Rubin, An Improved ELISA for Anti-Native DNA by Elimination of Interference by Anti-Histone Antibodies; J. Immunol, Methods, at 359 (1983) discusses an enzyme linked immunosorbentassay for antibodies to native DNA in which methylated bovine serum albumin (mBSA) was used to link DNA antigen (which had undergone digestion with S.sub.1 nuclease to polystyrene. After immobilizing DNA to the kit a gelatin was incubated in the wells to avoid problems with nonspecific binding. Thereafter, the microwells could be stored until use, however, prior to use the immobilized DNA in the wells must be re-digested with S.sub.1 nuclease.
The final additional step requiring S.sub.1 nuclease re-digestion prior to use of the plate suggests that the ssDNA present in stored microtiter plates has reached an unacceptable level and the kit lacks stability.
The Rubin coating method was compared with the present invention and the results showed that the present invention has significantly better stability, specificity and sensitivity than this kit. Factors that may contribute to the results obtained by the present invention includes the order in which the plates are coated, the blocker used and the solution the blocking agent is placed in, the lack of Tween 20, the incubation times and temperatures employed for the various coating step.
Specifically, addressing some of the above listed factors it is believed that coating the blocker after the immobilized dsDNA is treated with S.sub.1 nuclease permits less nonspecific binding than does coating the blocker prior to treating the dsDNA with S.sub.1 nuclease, because the treatment of S.sub.1 nuclease can lead to open binding sites on the support which are not blocked. Furthermore, the gelatin blocker used by Rubin is not believed to block as efficiently as a casein blocker used in the present invention.
Combining the teachings of Rubin's precoating method above with a casein blocker, the use of casein as a general blocking agent is suggested in an article by Robert F. Vogt et al, 1987 J. Immunol. Methods 101, 43 still resulted in significantly lower stability, specificity and sensitivity results than the results obtained by the present invention. Again, the similar factors are believed to contribute to these results. Specifically, the solution that the blocker is placed in in the present invention is adapted to smoothly coat the wells uniformly and also may increase the stability of the precoated wells, unlike the blocker material suggested by Vogt. Furthermore, in development of the present invention, certain experimental results may suggest that using Tween 20 may adversely effect the test kit results and thus unlike the Rubin and Rubin-Vogt pre-coating procedures, Tween 20 is not employed in the present invention.
Furthermore, performance of an assay on serum suspected of containing anti-dsDNA antibodies can be performed in less then one hour and forty-five minutes which is significantly less time than required by the Rubin protocol.